Phosphorus containing cyclic nucleotides

ABSTRACT

The invention relates to a compound of formula (I)Wherein:B is adenine, guanine or hypoxanthineZ is hydrogen or a negative chargeR is -[CH2CH(R1)-O]n-R2, -CH2CH2X, in whichR1 is hydrogen or (C1-C6) alkylR2 is hydrogen or (C1-C6) alkyln is a number from 1 to 6X is OH, F, NR3R4R3 and R4 are independently from each other hydrogen or (C1-C6) alkyl and to methods of enzymatically treating these compounds with biocatalysts having cyclic phosphodiesterase activity.

This application is a divisional of copending patent application Ser.No. 09/167,191, filed Oct. 6, 1998 now U.S. Pat. No. 6,147,211.

Antisense therapy requires the use of modified nucleotides in order toblock gene expression by preventing messenger RNA (mRNA) translationinto active proteins. The criteria requested for optimal activity of anantisense oligonucleotide are resistance to endo- and exonucleases,affinity and specificity for the targeted mRNA sequence as well as easyuptake by the concerned cells, and finally destruction or inactivationof the mRNA. Certain chemical modifications of the nucleotides or of thephosphate backbone can allow such biological activity and severalapproaches have been used to this end.

The first-generation antisense oligonucleotides consist of DNA in whicha sulfur atom has been incorporated in replacement of an equatorialoxygen atom in the phosphate backbone. This PS backbone confers a betterresistance to nucleases, although it lowers the affinity of theoligonucleotide for its target. The mechanism of action involves RNaseH, an enzyme that specifically degrades the RNA in a DNA:RNA complex,therefore destroying the mRNA. The second-generation strategies usemixtures of modified oligoribonucleotides and oligodeoxyribonucleotidesin chimeric molecules. The part of the molecule composed ofribonucleotides is then responsible for the affinity for the targetedsequence, whereas the part containing the deoxyribonucleotides acts as asubstrate for RNase H which cleaves the targeted mRNA. Theribonucleotides are modified at the C2′ position of the sugar wheredifferent substituents can be attached.

Chemical synthesis of the 2′-modified nucleotides is currently veryexpensive and, as the demand for such compound is expected to soar inthe near future, their biosynthesis by an appropriate microorganismwould markedly lower the cost of their production, especially if it ispossible to start with cheap compounds available in large amounts.

Cyclic nucleotides are essential elements in many biochemical processesof the eukaryotic and prokaryotic cell cycles, particularly adenosine3′,5′ cyclic monophosphate and guanosine 3′,5′ cyclic monophosphate.These compounds constitute the most adequate starting material for thepurpose of the present invention.

It is known that such natural cyclic nucleotides could be transformed totheir corresponding 5′-nucleoside monophosphates by 3′,5′-cyclicnucleotide phosphodiesterases e.g., from Vibrio fischeri (WO 94/16063,Journal of Bacteriology, Vol. 175, No. 15, p. 4615-4624). Furthermorethe conversion of 2′ O-methyl- and 2′ O-ethyl- nucleoside 3′,5′-cyclicphosphates to the corresponding 5′-phosphates by enzymatic hydrolysishas been published (Biochemistry, Vol. 11, No. 26, 1972, p. 4931-4937).

There is still a need for a process which allows the synthesis ofnucleotides with more complex 2′-substituents.

Surprisingly an enzymatic process has now been found to producenucleosides with such more complex 2′-substituents.

The invention relates to a process comprising treatment of compounds ofFormula (I)

wherein

B is adenine, guanine or hypoxanthine

Z is hydrogen or a negative charge

R is —[CH₂CH(R¹)—O]_(n)—R², —CH₂CH₂X, in which

R¹ is hydrogen or (C₁-C₆) alkyl

R² is hydrogen or (C₁-C₆) alkyl

n is a number from 1 to 6

X is OH, F, NR³R⁴

R³ and R⁴ are independently from each other hydrogen or (C₁-C₆) alkylwith a biocatalyst having cyclic phosphodiesterase activity.

The product of such process may in a second step be hydrolysed to acompound of Formula (II).

wherein B and R have the same meaning as defined above. This hydrolysiscould be done chemically or enzymatically.

Biocatalyst having cyclic phosphodiesterase activity may be for thepurpose of this invention a purified enzyme, from original orrecombinant source, a partially purified enzyme, a crude cell extract,enzymes immobilized or caught in partially broken cells or cell debris.The biocatalyst may be used in solution, suspension or in immobilizedform (matrix bound) according to routine processes, e.g. batch orcontinuous, known in the art. Source for the biocatalyst may be everyorganism, e.g. animal organs, microorganisms like fungi or bacteria,which comprise an enzyme suitable for being used as biocatalyst in theabove process. Whether a microorganism contains such enzyme can easilybe evaluated by a test which comprises the comparative growth of anorganism on a medium containing only salts and Tris buffer (minimalmedium, described in Dunlap et al. J. Gen. Microbiol. 1992, 138,115-123) and in parallel on the same medium supplemented with 1 to 5 mMof compound of formula (I). Any microorganism containing anextracellular or periplasmic (for gram-negative bacteria) enzyme able tohydrolyse the substrate will grow on the supplemented medium. Theminimal medium serves as a negative control differentiating betweenmicroorganisms able to grow on Tris or on cAMP only, as some microbesare known to be able to utilise Tris as a nutrient. For microorganismpossessing a cytoplasmic phophodiesterase (as it is the case for manybacteria, e.g., Escherichia coli or Salmonella typhimurium), growth onnutrient agar is necessary. The cell disruption supernatant has to beassayed as the cytoplasmic membrane is known to be fairly impermeable tocyclic nucleotides.

Accordingly the present invention also comprises new enzymes which canbe identified in the above tests.

A preferred embodiment of the invention is the use of the gram negativebacterium Serratia marcescens, particularly the use of strain DSM 30121as the source of biocatalyst having cyclic nucleotide phophodiesteraseactivity. Such biocatalyst having cyclic nucleotide phosphodiesteraseactivity is used as a purified enzyme, a partially purified enzyme or asa crude cell extract.

For the specific cleavage of the 3′,5′ nucleoside cyclic monophosphateto the 5′-monophosphate only the enzymatic cleavage is selective andtherefore clearly superior to chemical hydrolysis.

Furthermore the invention relates to compounds of formula (I)

wherein

B is adenine, guanine or hypoxanthine

Z is hydrogen or a negative charge

R is —[CH₂CH(R¹)—O]_(n)—R², —CH₂CH₂X, in which

R¹ is hydrogen or (C₁-C₆) alkyl

R² is hydrogen or (C₁-C₆) alkyl

n is a number from 1 to 6

X is OH, F, NR³R⁴

R³ and R⁴ are independently from each other hydrogen or (C₁-C₆) alkyl

Preferred are compounds of formula (I) wherein R is CH₂CH₂OR²,preferably CH₂CH₂OCH₃ further preferred are compounds wherein B isadenine.

The compounds of formula (I) could be synthesized by reacting thecorresponding nucleoside 3′,5′ cyclic monophosphates with an alkylationreagent, for example R-halogene, especially R-Br or R-I.

Furthermore the invention comprises the use of a biocatalyst havingcyclic phosphodiesterase activity for the preparation of compounds offormula (II).

The following examples shall illustrate the invention but not restrictit.

EXAMPLES Compounds Example 1 Preparation of2′-O(2-Methoxyethyl)-adenosine 3′,5′-cyclic-phosphate (2′MOE cAMP)

5.0 g (15.2 mmol) adenosine-3′:5′-cyclic phosphate are dissolved in 60ml DMSO. 1.28 g (22.8 mmol) powdered KOH and 1.80 ml (20 mmol)2-chloroethylmethylether are added. The mixture is stirred for 30 h at70° C., after 21 h additional 0.64 g KOH and 0.9 ml2-chloroethylmethylether are added. After cooling the mixture isevaporated and the residue is purified by chromatography (SiO₂,CH₂Cl₂/MeOH/H₂O 60:30:4). Obtained are beige crystals, Mp>250° C.

³¹P-NMR(D₂O): −0.759 ppm MS: 386(M-H)⁻

Example 2 Preparation of Sodium2′-O(2-Methoxyethyl)-guanosine-3′,5′-cyclic monophosphate (2′MOE cGMP)

0.40 g (1.09 mmol) sodium guanosine-3′:5′-cyclic monophosphate aredissolved in 4 ml DMSO. 0.73 g powdered KOH and 0.18 g (1.31 mmol)2-bromoethylmethylether are added. The mixture is stirred for 30 h at70° C. After cooling the mixture is evaporated and the residue ispurified by chromatography (SiO₂, CH₂Cl₂/MeOH/H₂O 60:30:4). Obtained arecolourless crystals,

³¹P-NMR(D₂O): −3.59 ppm MS: 402(M-Na)⁻

The substrates used for enzyme activity assays are dissolved inphosphate buffer pH 7.0 to the required concentration.

Fermentation Conditions

Serratia marcescens strain DSM 30121 is grown for 48 hrs in Petri dishescontaining Luria-Bertani [LB] Agar (1% Tryptone [Merck 07213]; 0.5%Yeast Extract [Fluka 70161]; 86 mM NaCl [Fluka 71382]; 50 mM Tris-HCl,pH 7.5 [Merck 8382]; 1.5% Bacto-Agar [Difco 0140-01]) at 30° C. (MemmertIncubator, Haska AG, Bern, Switzerland). One single colony issubsequently used to inoculate 25 ml of LB Broth contained in a 100-mlshake-flask. This culture is then incubated at 30° C. and shaken at 200rpm (Infors HT Shaker, Infors AG, Bottmingen, Switzerland) for another48 hrs.

Activity Assay

After incubation, the whole volume of the culture is centrifuged at 9000rpm for 20 minutes at ca. 4° C. (DuPont Instruments Sorvall® RC-5BRefrigerated Superspeed Centrifuge, Digitana AG, Horgen, Switzerland;rotor SS-34).

The supernatant (fraction A) is recuperated and 360 μl are placed in anEppendorf tube. Fourty μl of a 100 mM solution of substrate are thenadded to the reaction mixture and incubated overnight at 30° C.

The pellet is resuspended in 1 ml of distilled water and 40 μl of thiswhole-cell suspension (fraction B) are placed in an Eppendorf tube towhich 360 μl of a 10 mM solution of the substrate are added. Theincubation conditions are the same as for the supernatant fraction.

Six hundred μl of the resuspended 9000 rpm pellet are placed in anEppendorf tube containing 1.2 g of glass beads (Roth AG, Karlsruhe,Germany). The cells are subsequently broken in a mechanical celldisruptor (Retsch Mill, Schieritz & Hauenstein AG, Arlesheim,Switzerland) set at 100% for 10 minutes. The mixture is thencentrifugated at 14 000 rpm (Bench Centrifuge Biofuge 15, Heraeus AG,Zürich, Switzerland) and 40 μl of the supernatant (fraction C) is placedin an Eppendorf tube with 360 μl of a 10 mM solution of the substrate.Incubation is realised in the same manner as preceedingly.

After incubation, the three different fractions (fraction A:supernatant,fraction B: whole-cell suspension and fraction C: disrupted cellssupernatant) are treated the same way. The reaction is stopped by theaddition of 400 μl of methanol (Fluka 65543). After 5 minutes ofextraction at room temperature, the tubes are centrifugated at 14 000rpm for 5 minutes (Bench Centrifuge Biofuge 15) and the supernatanttransfered to HPLC tubes for analysis.

Blanks are made with the substrate solution being replaced by anequivalent volume of phosphate buffer pH 7.0.

Determination of Activity

The activity is qualitatively determined by observing the appearance ofthe substrate and product(s) of the activity assay as peaks on a HPLCprofile. The detection of peaks is compared to standards containing onlyone compound (cAMP, 3′-AMP, 5′-AMP, adenosine, and their 2′-modifiedcounterparts). A peak showing on the assay's chromatogramme at the sameor at a very close retention time as that of a standard, and moreoverabsent from the blank, is considered to be the same compound as thestandard with a minimum risk of error.

The chromatographic system used for the detection of nucleotides in theactivity assays is composed of the following elements (all fromMerck-Hitachi): AS-2000 autosampler, L-6210 pump (flow rate=0.8ml.min⁻¹), L-4000 UV-vis detector (detection wavelength set at 254 nm)and D-2500 integrator. The column is a Merck Lichrocart® 125-4 packedwith Lichrospher® 100 RP-18 (5 μm). A precolumn Merck Lichrocart® 4-4 isalso used upstream from the column.

The solvent system is composed of 10 mm phosphate buffer, pH 4.0(solvent A) and Methanol (solvent B) and two different gradients areapplied depending on whether natural or modified nucleotides are usedfor the assay:

Gradient for natural Gradient for modified nucleotides: nucleotides:Time Time (min) % A % B (min) % A % B 0.0 95 5 0.0 95 5 9.0 95 5 12.0 8515 15.0 80 20 19.0 40 60 17.0 0 100 19.5 0 100 19.0 0 100 26.0 0 10019.5 95 5 27.0 95 5 25.0 95 5 30.0 95 5

The HPLC profiles obtained for the activity assays are compared withprofiles of standards in order to identify the peaks.

Results

Intact cells (fraction B) of Serratia marcescens are found to degradeover 99% of added CAMP, whereas 2′MOE CAMP is about 85% degraded, thepresence of 2′MOE adenosine was also detected in the assay. Disruptedcells (fraction C) are able to hydrolyse about 99% or more of both CAMPand 2′ MOE CAMP. The cell free culture supernatant (fraction A) showshydrolysis of CAMP and 2′MOE CAMP in proportions of about 97% and about50% respectively. This fraction is also able to degrade cIMP (ca.85-89%) and cCMP (ca. 10%).

Chemical Cleavage of the 5′-monophosphates

1 ml acetic acid and 3 mg CeSO₄ are added to a solution of 7.3 mg of theammonium salt of 2′-O(2-Methoxyethyl)-adenosine-5′-monophosphate in 30ml H₂O. After 2 days the mixture is evaporated and chromatographed(SiO₂, MeOH/AcOH 1:4). 2′O(2-Methoxyethyl)-adenosine is obtained as awhite powder.

What is claimed is:
 1. A compound of formula (I)

wherein: B is adenine, guanine or hypoxanthine; Z is hydrogen or anegative charge; and R is −[CH₂CH(R¹)—O]_(n)—R² or —CH₂CH₂X, in whichwherein R¹ is hydrogen or (C₁-C₆) alkyl, R² is hydrogen or (C₁-C₆)alkyl, n is a number from 1 to 6, X is OH, F, NR³R⁴, and R³ and R⁴ areindependently from each other hydrogen or (C₁-C₆) alkyl; or a saltthereof when Z is a negative charge.
 2. The compound of claim 1 whereinR is CH₂CH₂OR².
 3. The compound of claim 1 wherein B is adenine.
 4. Thecompound of claim 2 wherein R is CH₂CH₂OCH₃.